Human immunodeficiency virus (HIV-1) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). The global HIV-1 pandemic (~35 million people infected) is sustained by 2-3 million new infections annually. Changing the course of this pandemic requires prevention of HIV-1 transmission, most of which occurs sexually. In the absence of an effective vaccine, modalities that block sexual HIV-1 transmission are desperately needed. Recently, the use of specific antiretroviral drugs has been investigated and shown to partially protect at-risk sex partners from HIV-1 infection. However, partial efficacy, drug side effects and the emergence of drug-resistant viruses limit the general applicability of these particular agents as prophylactic measures. New broadly active antiviral agents that can be used as topical microbicides could remedy these deficiencies. The HIV-1 envelope glycoproteins (Envs), which mediate virus entry into target cells, represent attractive targets for such prophylactic agents. The HIV-1 Envs are exposed on the viral surface, are accessible to water-soluble inhibitors and are present in low numbers on each virion. Env inhibitors used as microbicides need not be systemically absorbed or taken up by host cells, limiting potential toxicity. Conserved elements of Env mediate receptor binding, conformational changes, and membrane fusion, providing several potential targets for inhibition. Although some HIV-1 entry inhibitors have been identified, drug-resistant HIV-1 variants either exist naturally o develop during treatment. We have devised a screening strategy to identify new broad-range inhibitors of HIV-1 entry. In our screening assay, HIV-1 Env function leads to the fusion of Env-expressing cells with cells expressing the viral receptors. This cell-cell fusion assay and a specificity control assay will be used in parallel in a primary screen of the NIH small-molecule library. The highest-ranked compounds will be validated by a secondary screen that assesses reproducibility, dose-response, breadth and specificity. Tertiary assays involving single-round replication of viruses with multiple HIV-1 and other retroviral envelope glycoproteins can be performed at modest throughput and will provide information on potency, breadth and specificity. Additional tertiary assays to evaluate mechanism of action are well-established in the principal investigator's laboratory, and will be applied to selected compounds. Specific and broad-range inhibitors will be modified to improve potency while retaining breadth. The compounds identified in this study could potentially be useful as prophylactic microbicides, as treatments for already infected individuals, and as probes to understand the complex, multi-step process of HIV-1 entry.